Rapid display method in translational synthesis of peptide

ABSTRACT

Provided are linkers suitable for preparing a conjugate of a nucleic acid and a peptide as a translation product thereof in a reconstituted cell-free translation system in genotype-phenotype mapping (display methods), said linkers comprising a single-stranded structure region having a side chain base pairing with the base at the 3′-end of an mRNA at one end and a peptidyl acceptor region containing an amino acid attached to an oligo RNA consisting of a nucleotide sequence of ACCA via an ester bond at the other end, characterized in that the ester bond is formed by using an artificial RNA catalyst. Also provided are display methods using [mRNA]-[linker]-[peptide] conjugates assembled via such linkers.

TECHNICAL FIELD

The present invention relates to a novel method used for preparing aconjugate of a cDNA or mRNA and a peptide or protein translatedtherefrom in genotype-phenotype mapping (display systems). This methodwas designated as RAPID display method by us. This method is suitablefor screening peptide aptamers as potential drug candidates from unusualpeptide libraries constructed using the previously reported flexizymesystem (or RAPID system: Random Peptide Integrated Discovery system).

BACKGROUND ART

Technologies for mapping genotype and phenotype, also known as displaymethods, were born as tools for evolutionary molecular engineering, andinclude various known methods such as mRNA display (“in vitro virus”,Nemoto N. et al. FEBS Lett. 414, 405-408 (1997), InternationalPublication WO98/16636; or “RNA-peptide fusions”, Roberts, R. W. &Szostak, J. W., Proc. Natl. Acad. Sci. USA., 94, 12297-12302 (1997),International Publication WO98/31700), STABLE (non-covalent DNAdisplay), microbead/droplet display, covalent DNA display, phagedisplay, ribosome display, etc. Display methods are useful for selectinggenetic information of a polypeptide having a specific function becausea gene corresponding to a functional peptide or protein moleculeselected from a library is conjugated to such a molecule so that thesequence thereof can be readily read.

mRNA display is a technique for linking genotype and phenotype bycovalently coupling an mRNA as genotype and a peptide molecule asphenotype using a cell-free translation system (in vitro proteinsynthesis system), and currently applied by coupling a synthesizedpeptide molecule and an mRNA encoding it via puromycin, which is ananalogue of the 3′ end of a tyrosyl-tRNA.

In mRNA display, an mRNA containing puromycin preliminarily attached toits 3′ end via a suitable linker is introduced into a cell-freetranslation system to synthesize a peptide from the mRNA so that thepuromycin is fused to the C-terminus of a growing peptide chain as asubstrate for peptidyl transfer reaction on a ribosome and thetranslated peptide molecule is fused to the mRNA via the puromycin (FIG.1A). The linker is inserted between the mRNA and the puromycin mainlyfor the purpose of efficiently incorporating the puromycin into the Asite of the ribosome. Puromycin is characterized in that theadenosine-like moiety and the amino acid (tyrosine)-like moiety form anamide bond rather than an ester bond, unlike the 3′ end of anaminoacyl-tRNA (FIG. 1B). Thus, the conjugate of the puromycin and thepeptide fused to each other on the ribosome is resistant to hydrolysisand stable.

In mRNA display, it is necessary to attach puromycin to the 3′ end ofthe mRNA in advance outside the cell-free translation system in order tocouple the mRNA and the translation product via the puromycin. Thisattachment takes place by either first preparing a puromycin-conjugatedlinker having a spacer consisting of a linear polymer synthesized at the5′ end from puromycin and then fusing the linker to the 3′ end of themRNA or conjugating a spacer to the 3′ end of the mRNA and then fusingthe puromycin to the conjugate. In either method, the linear polymerspacer typically contains a phosphate group or nucleotide at an end, andthe linkage between the 3′ end of the mRNA and the 5′ end of the linkeris a covalent bond via the phosphate group. This covalent bond is formedby a reaction using an RNA ligase or DNA ligase or a standard organicchemistry reaction.

CITATION LIST Patent Documents

-   Patent document 1: Japanese Patent No. 3683282 (International    Publication WO98/16636)-   Patent document 2: Japanese Patent No. 3683902-   Patent document 3: Japanese Patent No. 3692542 (International    Publication WO98/31700)

Non-Patent Documents

-   Non-patent document 1: Nemoto N. et al. FEBS Lett. 414, 405-408    (1997)-   Non-patent document 2: Roberts, R. W. & Szostak, J. W., Proc. Natl.    Acad. Sci. USA., 94, 12297-12302 (1997)

SUMMARY OF INVENTION Technical Problems

In known mRNA display methods, an mRNA template having puromycin at the3′ end is added to a cell-free translation system using wheat germextract or rabbit reticulocyte lysate to translate it into a peptide.Thus, it is necessary to carry out transcription from DNA into mRNA andfusion reaction between mRNA and puromycin in advance outside thetranslation system.

Recently, reconstituted cell-free translation systems were developed byindividually purifying and mixing elements necessary for translation insystems using E. coli ribosomes (H. F. Kung, B. Redfield, B. V.Treadwell, B. Eskin, C. Spears and H. Weissbach (1977) “DNA-directed invitro synthesis of beta-galactosidase. Studies with purified factors”The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894; M. C.Gonza, C. Cunningham and R. M. Green (1985) “Isolation and point ofaction of a factor from Escherichia coli required to reconstructtranslation” Proceeding of National Academy of Sciences of the UnitedStates of America Vol. 82, 1648-1652; M. Y. Pavlov and M. Ehrenberg(1996) “Rate of translation of natural mRNAs in an optimized in vitrosystem” Archives of Biochemistry and Biophysics Vol. 328, No. 1, 9-16;Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawaand T. Ueda (2001) “Cell-free translation reconstituted with purifiedcomponents” Nature Biotechnology Vol. 19, No. 8, 751-755; H. Ohashi, Y.Shimizu, B. W. Ying, and T. Ueda (2007) “Efficient protein selectionbased on ribosome display system with purified components” Biochemicaland Biophysical Research Communications Vol. 352, No. 1, 270-276). Thereconstituted cell-free translation systems are freed of elementsirrelevant to translation by fractionating a cell-free translationsystem based on an E. coli extract and reassembling fractions so thatinclusion of inhibitors such as nucleases and proteases can be preventedmore easily than in conventional cell-free translation systems usingcell extracts. Transcription from DNA and translation can also besimultaneously performed by adding elements necessary for transcriptionreaction.

If such a cell-free coupled transcription-translation system is used,mRNA synthesis by transcription from template DNA can take place in thesame system as translation reaction. Moreover, if complex formationbetween an mRNA and a linker molecule could also take place in the samesystem, transcription of cDNA to preparation of an mRNA-peptide fusioncould be accomplished in one pot (in the same reaction vessel), unlikeconventional mRNA display methods. The first object of the presentinvention is to provide a display method taking advantage of such areconstituted cell-free translation system capable of controllingcomponents of the reaction system.

Further, the second object of the present invention is to make itpossible to construct an mRNA or cDNA library presenting unusualpeptides by combining such a display method with a previously reportedtechnique for synthesizing a unusual peptide using a ribozyme capable ofcatalyzing the synthesis of an acylated tRNA (flexizyme).

Solution to Problems

The RAPID display method of the present invention made it possible tocompletely accomplish transcription, translation and linker-mRNA complexformation followed by linkage between the peptide and the linker in asingle translation system by modifying mRNA display to replace thepuromycin-conjugated linker by a linker molecule now developed by us andfurther optimizing the reconstituted cell-free translation system.

As compared with conventional mRNA display methods, the RAPID displaymethod mainly has the following features.

(a) The 3′ end of the linker molecule has a structure in which an aminoacid is attached to adenosine via an ester (i.e., aminoacylated) ratherthan puromycin.(b) Aminoacylation reaction is mediated by an artificial RNA catalyst(ribozyme).(c) A reconstituted cell-free translation system is used.(d) The fusion between the linker and an mRNA is made by complexformation based on hybridization in a translation system rather thanligation.(e) Transcription, translation and complex formation with the linker canbe performed in a single translation reaction vessel.

Moreover, unusual peptides can also be presented as phenotypes byapplying techniques for synthesizing unusual peptides by translation inthe same translation system. Acylation reaction for charging a tRNA witha non-proteinogenic amino acid or hydroxy acid, which is a constituentunit of a unusual peptide, is also mediated by an artificial RNAcatalyst (ribozyme).

The present invention is summarized as follows.

(1) A linker used for preparing a conjugate in which an mRNA and apeptide as a translation product thereof are coupled via the linker in areconstituted in vitro protein synthesis system, said linker comprising:a single-stranded structure region having side chain bases pairing withthe bases at the 3′-end of the mRNA at one end of the linker, anda peptidyl acceptor region having a group capable of binding to thetranslation product by peptidyl transfer reaction at the other end ofthe linker,wherein the peptidyl acceptor region has a structure containing an aminoacid attached to an oligo RNA consisting of a nucleotide sequence ofACCA via an ester bond; andsaid ester bond is formed by an aminoacylation reaction using anartificial RNA catalyst.(2) The linker as defined in (1) above wherein the single-strandedstructure region and the peptidyl acceptor region are connected via apolyethylene glycol moiety.(3) The linker as defined in (1) or (2) above wherein thesingle-stranded structure region consists of a single-stranded DNA.(4) The linker as defined in any one of (1)-(3) above wherein theartificial RNA catalyst used in the aminoacylation reaction has achemical structure consisting of any one of the RNA sequences below:

(SEQ ID NO: 3) GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU(SEQ ID NO: 4) GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU(SEQ ID NO: 5) GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGGU(SEQ ID NO: 19) GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGGU.(5) A process for preparing an [mRNA]-[linker]-[peptide] conjugate inwhich an mRNA and a peptide as a translation product thereof are coupledvia the linker as defined in any one of (1)-(4) above, said processcomprising the steps of:preparing the linker as defined in any one of (1)-(4) above;synthesizing an mRNA having a sequence capable of hybridizing with thebase sequence ofthe single-stranded structure region of the linker downstream of asequence encoding a peptide; andcontacting the linker with the mRNA and translating the mRNA into thepeptide in a reconstituted in vitro protein synthesis reaction solution.(6) A process for preparing an [mRNA]-[linker]-[peptide] conjugate inwhich an mRNA and a peptide as a translation product thereof are coupledvia the linker as defined in any one of (1)-(4) above, said processcomprising the steps of:preparing the linker as defined in any one of (1)-(4) above,synthesizing a template DNA for an mRNA having a sequence capable ofhybridizing with the base sequence of the single-stranded structureregion of the linker downstream of a sequence encoding a peptide, andintroducing the linker and the DNA into a reconstituted in vitro proteinsynthesis reaction solution, thereby performing transcription from theDNA into the mRNA and translation into the peptide as well as complexformation between the linker and the mRNA.(7) The process as defined in (5) or (6) above wherein the reconstitutedin vitro protein synthesis reaction solution contains a tRNA chargedwith a non-proteinogenic amino acid or hydroxy acid, whereby thetranslated peptide constitutes a unusual peptide.(8) A library comprising [mRNA]-[linker]-[unusual peptide] conjugatesprepared by the process as defined in (7) above.(9) A method for selecting a peptide aptamer that binds to a targetsubstance from a library of [mRNA]-[linker]-[peptide] conjugates inwhich each mRNA and a peptide as a translation product thereof arecoupled via the linker as defined in any one of (1)-(4) above, saidmethod comprising the steps of:preparing the linker as defined in any one of (1)-(4) above;preparing an mRNA library comprising mRNAs each having a sequencecapable ofhybridizing with the base sequence of the single-stranded structureregion of the linker downstream of a sequence encoding a random peptidesequence;contacting the linker with the mRNA library and performing translationinto the peptide in a reconstituted in vitro protein synthesis reactionsolution, thereby preparing an [mRNA]-[linker]-[peptide] conjugatelibrary;contacting the target substance with the [mRNA]-[linker]-[peptide]conjugate library; andselecting a conjugate presenting the peptide bound to the targetsubstance.(10) The method as defined in (9) above wherein the target substance hasbeen biotinylated.(11) The method as defined in (9) or (10) above wherein thereconstituted in vitro protein synthesis reaction solution contains atRNA charged with a non-proteinogenic amino acid or hydroxy acid,whereby the translated peptide constitutes a unusual peptide.(12) A process for preparing the linker as defined in (2) above, saidprocess comprising the steps of:synthesizing a chimeric oligonucleotide consisting of thesingle-stranded structure region and an oligo RNA of a sequence of ACCAconnected via a polyethylene glycol moiety; andattaching an amino acid to adenosine at the 3′ end of the chimericoligonucleotide via an ester bond by a reaction using an artificial RNAcatalyst,thereby preparing a linker consisting of the single-stranded structureregion and the peptidyl acceptor region connected via the polyethyleneglycol moiety.(13) The process as defined in (12) above wherein the artificial RNAcatalyst has a chemical structure consisting of any one of the RNAsequences below:

(SEQ ID NO: 3) GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU(SEQ ID NO: 4) GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU(SEQ ID NO: 5) GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGGU(SEQ ID NO: 19) GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGGU.

Advantageous Effects of Invention

By using a linker molecule capable of binding to an mRNA in areconstituted cell-free translation reaction solution, an[mRNA]-[linker]-[peptide] conjugate can be prepared only by adding thelinker and a cDNA or mRNA to perform translation reaction in a singlereaction vessel.

Moreover, an [mRNA]-[linker]-[unusual peptide] conjugate presenting aunusual peptide synthesized by translation from sequence information ofa template nucleic acid molecule can be obtained by introducing a tRNAcharged with a non-proteinogenic amino acid or hydroxy acid into thesame reconstituted cell-free translation reaction solution.

Thus, a gene library of artificial peptide aptamers expected to improvein vivo stability and binding affinity for a target protein can besimply constructed by combining the RAPID display method with atechnique for synthesizing a unusual peptide by translation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A schematically shows the process in which a translated peptidemolecule is coupled to an mRNA via a puromycin-conjugated linker in mRNAdisplay.

FIG. 1B shows the structure of the peptidyl acceptor region of thelinker (puromycin-conjugated linker).

FIG. 2A schematically shows the process in which a translated peptidemolecule is coupled to an mRNA via an RAPID linker (e.g., anL-Phe-conjugated linker shown as an example) in the RAPID display methodof the present invention.

FIG. 2B shows the structure of the peptidyl acceptor region of thelinker (L-Phe-conjugated linker).

FIG. 3 shows that a linker molecule (an 21-ACCA) has been aminoacylatedby a flexizyme-catalyzed reaction (Example 1).

FIG. 4 shows the yields of complexes of a peptide aptamer (TNF-α-DW) andan mRNA when the mRNA was added to the reaction solution (Example 1).

FIG. 5 shows the yields of complexes of a peptide aptamer (TNF-α-DW) andan mRNA when a cDNA was added to the reaction solution (Example 1).

FIG. 6 shows the result confirming that the peptide-mRNA complexpresenting TNF-α-DW was recovered more efficiently than a control(EMP1SS) complex unbounded to the target (Example 2).

FIG. 7 shows changes in binding affinity with the number of rounds whena peptide aptamer was selected by repeating selection multiple timesusing a TNF-α-DW-spiked library and an 21-Phe linker (Example 3).

DETAILED DESCRIPTION OF THE INVENTION

1. Linker

The linker in the RAPID display method of the present invention connectsan mRNA and a peptide translated therefrom by binding to the 3′ end ofthe mRNA at one end and to the C-terminus of the peptide at the otherend in the same manner as in known mRNA display methods.

However, the linker in the RAPID display method of the present inventiondiffers in the structure of both ends from those used in known mRNAdisplay methods. The linker used in the RAPID display method of thepresent invention is herein sometimes referred to as “RAPID linker”.

First, the region at one end of the linker binding to the C-terminus ofa peptide is explained. This region is herein sometimes referred to as“peptidyl acceptor” or simply “acceptor”. Thus, the term “peptidylacceptor” refers to a molecule having a structure capable of binding toa peptide growing by peptidyl transfer reaction on a ribosome(peptidyl-tRNA). The peptidyl acceptor may refer to a region located atan end of a linker or may refer to a whole structure including a linker.For example, the peptidyl acceptor in known mRNA display methods ispuromycin located at one end of a linker or a puromycin-conjugatedlinker as a whole structure including a linker.

The RAPID linker of the present invention is characterized by thestructure and the preparation process of the peptidyl acceptor.

In the RAPID display method, a linker having a sequence consisting of a4-residue ribonucleotide ACCA is synthesized at the 3′ end, and then agiven amino acid is attached to adenosine at the 3′ end, therebyconferring a structure as peptidyl acceptor on the linker. Duringpeptide elongation reaction on a ribosome, the amino acid attached tothe end of the linker accepts the C-terminus of the peptide of thepeptidyl-tRNA and binds to the peptide. The structure in which an aminoacid is attached to the RNA sequence ACCA via an ester bond is hereinreferred to as “peptidyl acceptor region”.

The peptidyl acceptor in known mRNA display methods is puromycin thathas an aminonucleoside structure in which a ribose in the adenosine-likemoiety and an amino acid are linked via an amide bond. In the RAPIDdisplay method of the present invention, however, an amino acid isattached to the 3′-O of ribose via an ester bond. In other words, thepeptidyl acceptor in the RAPID display of the present invention has anucleoside structure similar to that of natural aminoacyl-tRNA. See FIG.2B showing the structure of a linker to which L-phenylalanine isattached as an example of such a peptidyl acceptor (L-Phe-conjugatedlinker) in comparison with FIG. 1B (puromycin-conjugated linker). In thepresent invention, the peptidyl acceptor shows an incorporationefficiency comparable to or higher than that of puromycin by adopting astructure closer to that of the natural acceptor.

The formation of a bond between the peptidyl acceptor and the C-terminusof the peptide seems to occur by the proximity of the amino group of thepeptidyl acceptor incorporated into the A site to the ester bond at theC-terminus of the attached peptide of the peptidyl-tRNA in the P site inthe same manner as normal peptidyl transfer reaction in ribosomes. Thus,the covalent bond formed with the C-terminus of the peptide chain istypically an amide bond in the same manner as in mRNA display. It shouldbe noted that a linker having an unnatural (non-proteinogenic) aminoacid such as a D-amino acid or β (beta)-amino acid can also be used inthe RAPID display of the present invention by using an artificial RNAcatalyst (flexizyme) for the synthesis of the linker.

Next, binding to an mRNA at the other end of the linker is explained.

In the RAPID display method of the present invention, the 5′ end of thelinker and the 3′ end of a mRNA molecule forms a complex byhybridization based on base pairing. Thus, the 5′ end of the linkerassumes a single-stranded structure having a nucleic acid base in theside chain. This region in the RAPID linker is herein referred to as“single-stranded structure region”. Specific examples of single-strandedstructures having a nucleic acid base in the side chain includesingle-stranded DNAs, single-stranded RNAs, single-stranded PNAs(peptide nucleic acids), etc. The resulting complexes must be alsostably kept during peptide selection. As the complementarity between thenucleotide sequence of the single-stranded structure region of thelinker and the sequence of the 3′ end of the mRNA molecule increases,the efficiency of double-strand formation increases and stability alsoincreases. Stability also depends on the GC content, the saltconcentration of the reaction solution, and reaction temperature.Especially, this region desirably has a high GC content, specifically aGC content of 80% or more, preferably 85% or more. Specific examples ofsuch structures at the 5′ end of the linker include, but in any way arenot limited to, single-stranded DNAs consisting of 13-21 nucleotidesused in the Examples herein below having the nucleotide sequences:

5′-CTCCCGCCCCCCGTCC-3′ (SEQ ID NO: 1) 5′-CCCGCCTCCCGCCCCCCGTCC-3′.(SEQ ID NO: 2)

The rest of the linker excluding both ends is designed to have aflexible, hydrophilic and simple linear structure with less side chainsas a whole similarly to the structure of linkers used in known mRNAdisplay methods. Therefore, linear polymers including, for example,oligonucleotides such as single- or double-stranded DNA or RNA;polyalkylenes such as polyethylene; polyalkylene glycols such aspolyethylene glycol; polystyrenes; polysaccharides; or combinationsthereof can be appropriately selected and used. The linker preferablyhas a length of 100 angstroms or more, more preferably about 100-1000angstroms.

A specific non-limiting example of linkers that can be used in thepresent invention includes a chimeric DNA/RNA oligonucleotide comprisinga single-stranded structure region consisting of a single-stranded DNAhaving a high-GC content sequence and an RNA consisting of an ACCAsequence at the 3′ end wherein the DNA and RNA are connected via apolyethylene glycol moiety (PEG linker). For example, a typical exampleincludes [DNA]-[Spacer18]_(n)-rArCrCrA (wherein Spacer18 is hexaethyleneglycol, and n is an integer of 4-8) synthesized in Example 1.

2. Amino Acid Modification of the Linker

As indicated above, it is necessary to attach an amino acid to the oligoRNA moiety (ACCA sequence) at an end of the linker molecule in order toconfer a structure as a peptidyl acceptor on the linker. The presentinvention is characterized in that this attachment of an amino acidtakes place using an artificial RNA catalyst.

It is theoretically possible to attach an amino acid to the 3′-O of anoligo RNA synthesized by conventional chemical synthesis such assolid-phase synthesis via an ester, but it is practically impossible tointroduce the synthetic product into a cell-free translation systemafter post-treatment of synthesis reaction because it is highly reactiveand lacks stability. In the present invention, this problem is solved byaminoacylation reaction of the oligo RNA using a “flexizyme”, which isan artificial RNA catalyst developed as an aminoacyl-tRNA synthetase. Inthis method, aminoacylation reaction can be performed under mildconditions and the product can be introduced into a translation systemand used in it only after simple post-treatment. For details, see thefollowing documents:

-   H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology,    Vol. 10, 655-662;-   H. Murakami, D. Kourouklis, and H. Suga, (2003), Chemistry &    Biology, Vol. 10, 1077-1084;-   H. Murakami, A. Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3,    357-359;-   N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic &    Medicinal Chemistry Letters 19, 3892-3894; and-   JPA-2008-125396 or WO2007/066627.

Flexizymes are also known by designations such as dinitrobenzylflexizyme (dFx), enhanced flexizyme (eFx), amino flexizyme (aFx), etc.Flexizymes have the ability to catalyze aminoacylation of adenosine atthe 3′ end using a weakly activated amino acid as a substrate byrecognizing the carbonyl group with which the amino acid reacts, anaromatic ring in the side chain or leaving group of the amino acid, andan ACC-3′ sequence at the 3′ end of the linker. This is why an oligo RNAstructure consisting of an ACCA-3′ sequence is essential at the end ofthe RAPID linker. Flexizyme-mediated aminoacylation reaction proceedsonly by placing an amino acid substrate and a linker molecule having acognate oligo RNA moiety on ice in the presence of a flexizyme for about2 hours.

In the present invention, flexizymes having the sequences shown beloware suitably used.

Original flexizyme Fx (SEQ ID NO: 3)[GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU-3′, 45 nt]Enhanced flexizyme eFx (SEQ ID NO: 4)[5′-GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU- 3′, 45 nt])Dinitrobenzyl flexizyme dFx (SEQ ID NO: 5)[5′-GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGG U-3′, 46 nt]Amino flexizyme aFx (SEQ ID NO: 19)[5′-GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGG U-3′, 47 nt])

Flexizyme Fx is capable of catalyzing aminoacylation using amino acidsubstrates having a cyanomethyl leaving group and a side chain aromaticring (e.g., cyanomethyl esters of phenylalanine, tyrosine, etc.), whileflexizymes eFx, dFx, aFx are capable of catalyzing aminoacylation usingamino acid substrates having a 4-chlorobenzylthiol leaving group and anon-aromatic ring side chain in addition to the leaving groups and sidechains that can be used with flexizyme Fx. For details, see thedocuments cited above. By reacting an amino acid having such a structureand a linker in the presence of a flexizyme, therefore, a molecule inwhich the amino acid is attached via an ester bond to the 3′-hydroxylgroup on the ribose ring in adenosine at the 3′ end of the linker can beobtained. Amino acid substrates having any structure can be attached,including not only amino acids used in natural translation but alsonon-proteinogenic amino acids such as D-amino acids or β (beta)-aminoacids (i.e., other than L-amino acids normally found in naturallyoccurring proteins). Further, a hydroxycarboxylic acid instead of anamino acid can be attached to the 3′ end of the linker or incorporatedas a peptidyl acceptor into a ribosome.

3. Reaction in a Translation System

According to the RAPID display method, a linker is added to a cell-freetranslation system (also referred to as “in vitro protein synthesissystem”) along with a cDNA or mRNA and reacted for a predeterminedperiod, thereby allowing for translation from the mRNA and complexformation with the linker molecule followed by linkage between thepeptide and the linker.

The cell-free translation system used can be a conventionalreconstituted cell-free translation systems with appropriatemodifications, and transcription from DNA can also be performed in thesame system as used for translation if it contains a DNA-dependent RNApolymerase (preferably T7RNA polymerase).

The following reactions (a) to (d) can be performed in a single reactionvessel (one-pot), if it is a coupled transcription-translation synthesissystem:

(a) a reaction in which a DNA is transcribed into an mRNA;(b) a reaction in which the 3′ end of the mRNA forms a complex with thesingle-stranded structure region at an end of a linker viahybridization;(c) a reaction in which the mRNA is translated into a peptide;(d) a reaction in which the C-terminus of the translated peptide bindsto the peptidyl acceptor region at the other end of the linker via anamide bond;(e) a reaction in which a [peptide]-[linker]-[mRNA] complex is releasedfrom the ribosome.

Alternatively, a series of reactions starting from (b) are performed ina reconstituted cell-free translation system, when a preliminarilyprepared mRNA is added to the translation system along with a linker.

A reconstituted cell-free translation system should comprise purifiedribosomes, translation initiation factors, translation elongationfactors, an mRNA, an aminoacyl-tRNA, ATP or GTP used as a substrate,etc. (M. H. Schreier, B. Erni and T. Staehelin (1977) “Initiation ofmammalian protein synthesis. I. Purification and characterization ofseven initiation factors.” Journal of Molecular Biology, Vol. 116, No.4, 727-53. H. Trachsel, B. Emi, M. H. Schreier and T. Staehelin (1977)“Initiation of mammalian protein synthesis. II. The assembly of theinitiation complex with purified initiation factors.” Journal ofMolecular Biology, Vol. 116, No. 4, 755-67.). Among them, theaminoacyl-tRNA can be replaced by adding a tRNA, an aminoacyl-tRNAsynthetase and its substrate into the same reaction solution.Additionally, proteins or enzymes and their substrates such astranslation termination factors, ribosome recycling factors, creatinekinase, myokinase, nucleotide diphosphate kinase, pyrophosphatase can beadded to increase the efficiency or fidelity of translation reaction ascommon in conventional cell-free translation systems (P. C. Jelenc andC. G. Kurland (1979) “Nucleoside triphosphate regeneration decreases thefrequency of translation errors” Proceedings of the Natural AcademyScience of the United States of America Vol. 76, No. 7, 3174-3178). Whentranscription reaction is to be performed simultaneously withtranslation, a cDNA as well as T7 RNA polymerase and its substrate canbe added in place of mRNA.

In the present invention, a DNA or RNA having a necessary sequence isintroduced into a cell-free translation system comprising componentsoptimized for an intended purpose. The sequence of the DNA or RNA shouldbe such that a cDNA is transcribed into an mRNA and that translation ofthe mRNA starts in the synthesis system used. The full length of theregion encoding the amino acid sequence of a peptide should betranslated to the end, and a spacer sequence consisting of a peptide forconferring flexibility is also fused to the C-terminus of the translatedamino acid, immediately followed by a stop codon. For example, a peptidesequence (Cys- (Gly-Ser-)x3) and an amber codon (stop codon) immediatelydownstream of it are encoded. Additionally, the 3′ end of the mRNA has astructure capable of hybridizing with the single-stranded structureregion of a linker to form a double strand, so that the downstream ofthe coding region (immediately downstream of the stop codon) should havea sequence complementary to the sequence of the single-strandedstructure region. The sequence of this region (double strand-formingregion) is herein referred to as linker hybridization sequence.

Specifically, the cDNA or mRNA desirably contains the followingsequences:

(1) A promoter sequence compatible with the RNA polymerase used in thecDNA. TAATACGACTCACTATA (SEQ ID NO: 6) in the case of T7 promoter.

(2) A sequence encoding a relevant sequence upstream of a start codon.

When E. coli-derived ribosomes are used in a cell-free translationsystem, the gene for the SD sequence is included. This is also found inconventional protein synthesis. For example, GGGTTAACTTTAAGAAGGAGATATACAT (SEQ ID NO: 7): a modified sequence upstream of gene 10protein of T7 phage. The SD sequence is underlined.

(3) A sequence constituting an ORF [a sequence encoding an amino acidsequence having a spacer fused to the C-terminus of a peptide aptamer]of a variant gene library, beginning with a start codon (ATG).

Depending on the stop codon used here, its cognate release factor shouldbe removed from the cell-free translation system. A cell-freetranslation reaction solution is prepared by removing RF1 when TAG(amber codon) is used, or removing RF2 when TGA (opal codon) is used, orremoving both RF1 and RF2 when TAA (ochre codon) is used.

(4) A sequence encoding a double strand-forming region (linkerhybridization sequence).

The reconstituted cell-free translation reaction solution used in thepresent invention preferably contains components adapted for intendedpurposes. For example, the release factor cognate to the amber codon, apeptidyl-tRNA hydrolase (PTH), which is an enzyme cleavingpeptidyl-tRNA, and the like are removed in the Examples herein below.

Further, an acylated tRNA preliminarily charged with a desirednon-proteinogenic amino acid (or hydroxy acid) (i.e., having anactivated amino acid attached thereto) can be added to a reconstitutedcell-free translation system containing only limited natural aminoacids. By correlating the codons for excluded natural amino acids withthe anticodon of the tRNA acylated with a non-proteinogenic amino acid(or hydroxy acid), a peptide containing the non-proteinogenic amino acid(or hydroxy acid) can be synthesized by translation on a ribosome on thebasis of genetic information of the mRNA. Alternatively, a unusualpeptide containing no natural amino acid can also be synthesized bytranslation by adding an acylated tRNA charged with a non-proteinogenicamino acid (or hydroxy acid) to a reconstituted cell-free translationsystem containing no natural amino acid.

Acylated tRNAs charged with a non-proteinogenic amino acid (or hydroxyacid) can be prepared by using the artificial RNA catalysts “flexizymes”capable of catalyzing aminoacyl-tRNA synthesis as described above. Asindicated above, these artificial RNA catalysts are capable of chargingan amino acid having any side chain and also have the function ofrecognizing only a consensus sequence 5′-RCC-3′ (R=A or G) at the 3′ endof tRNAs to acylate the 3′ end of the tRNAs, and therefore, they can acton any tRNAs having different anticodons. Moreover, the recognition sitein an amino acid contains no substituent at the α position, so that notonly L-amino acids but also hydroxy acids (having a hydroxyl group atthe α-position), N-methylamino acids (having an N-methylamino acid atthe α-position), N-acylamino acids (having an N-acylamino group at theα-position), D-amino acids and the like can be used as substrates.Detailed description can be found in the documents about flexizymescited above as well as in Y. Goto, H. Suga (2009) “Translationinitiation with initiator tRNA charged with exotic peptides” Journal ofthe American Chemical Society, Vol. 131, No. 14, 5040-5041,WO2008/059823 entitled by “TRANSLATION AND SYNTHESIS OF POLYPEPTIDEHAVING NONNATIVE STRUCTURE AT N-TERMINUS AND APPLICATION THEREOF”, Gotoet al., ACS Chem. Biol., 2008, 3, 120-129, WO2008/117833 entitled by“PROCESS FOR SYNTHESIZING CYCLIC PEPTIDE COMPOUND”, etc.

A comprehensive technology for translation/synthesis, modification andscreening of peptides based on a core technology consisting of asynthesis system of unusual peptides (a concept including both kit andsynthesis method) using a tRNA acylated with a non-proteinogenic aminoacid or hydroxy acid via a “flexizyme” was designated by us as RAPIDsystem (Random Peptide Integrated Discovery system). The RAPID systemallows for the translation/synthesis of various unusual peptides as invitro translation products based on template mRNAs of relevantsequences. It should be understood from the foregoing description thatunusual peptides as used herein refer to polymers containing the varioussubstrates described above as their components and include anytranslation products that can be synthesized by the RAPID system otherthan twenty natural amino acids, including amino acids having variousside chains, β (beta)-amino acids, γ (gamma)-amino acids and δ(delta)-amino acids, D-amino acids, and derivatives having a structurein which an amino group or a carboxyl group on the amino acid backboneis substituted. Further, unusual peptides may have a backbone structureother than normal amide bonds. For example, unusual peptides alsoinclude depsipeptides consisting of amino and hydroxy acids, polyestersproduced by continuous condensation of hydroxy acids, peptidesmethylated at the nitrogen atom of the amide bond by introducing anN-methylamino acid, and peptides having various acyl groups (acetyl,pyroglutamic acid, fatty acids, etc.) at the N-terminus. Furthermore,cyclic peptides obtained by circularizing non-cyclic peptides consistingof an amino acid sequence bearing a pair of functional groups capable offorming a bond between them at opposite ends can also be synthesized bythe RAPID system (or cyclic N-methylpeptides can be obtained ifN-methylpeptides are used). Circularization may occur under theconditions of cell-free translation systems with a pair of somefunctional groups, as exemplified by a cyclic peptide circularized via athioether bond obtained by translation/synthesis of a peptide sequencebearing a chloroacetyl group and a cysteine group at opposite ends asshown in the Examples herein below.

The RAPID system allows for the synthesis of peptides having variousstructures only by changing template mRNAs because unusual peptides aresynthesized by ribosomal translation. If translation/synthesis isperformed using an mRNA (or corresponding DNA) containing a randomsequence, a random peptide library can be readily constructed. The RAPIDdisplay method of the present invention is suitably used to link unusualpeptides synthesized by the RAPID system to mRNAs representing theirgenotypes. The RAPID linker of the present invention is added along witha template cDNA or mRNA to a cell-free translation system optimized forthe synthesis of a unusual peptide and the mixture is reacted for apredetermined period, whereby the unusual peptide as the resultingtranslation product is coupled to the mRNA via the linker and presented.

Next, the linkage between the linker and the mRNA occurring in thecell-free translation system is explained. As described in the sectionof Background Art, it was necessary to ligate the linker and the mRNAoutside the translation system at a stage prior to translation reactionin known mRNA display methods. In contrast, the RAPID display method ofthe present invention is characterized in that complex formation betweenthe linker and the mRNA by hybridization can be carried out in thetranslation system.

With reference to FIG. 2, the process in which hybridization between anmRNA and a RAPID linker, synthesis of a peptide molecule and fusion ofthe linker to the C-terminus of the peptide take place by introducingthe mRNA along with the linker into a cell-free translation system isexplained.

As indicated above, the linkage between the mRNA and the linker resultsfrom the formation of a double strand via a hydrogen bond between thebase sequence of the single-stranded structure region of the linker anda complementary base sequence at the 3′ end of the mRNA. This linkage ismade to map the mRNA to the translated peptide molecule, so that an[mRNA]-[linker]-[peptide] complex formed during translation on aribosome must be also stably kept during the selection of the translatedpeptide.

By introducing an mRNA along with an RAPID linker into a cell-freetranslation system, a ribosome is located on the mRNA and translationreaction starts, whereby a peptide chain elongates and the terminatedpeptide chain binds to the amino acid of a peptidyl acceptor regionconsisting of [rACCA-amino acid] of the linker at the C-terminus anddissociates from tRNA. Without wishing to limit the concept of thepresent invention but for illustrative purposes only, we believe thatthe hybridization between the mRNA and the RAPID linker may occur at astage before the elongation reaction of a peptide chain starts if themRNA and the RAPID linker are introduced into the cell-free translationsystem at the same time. Alternatively, complex formation byhybridization between the mRNA and the RAPID linker on the ribosomeproperly occurs even when transcription from a cDNA and translationreaction take place first and then the linker is added into thetranslation reaction solution lacking termination factors and PTH. Then,a covalent linkage seems to occur between [rACCA-amino acid] at an endof the linker and the C-terminus of the peptide when this moietyaccidentally enters the ribosomal A site at the end of translation. Thereaction occurs with high efficiency because hybridization between themRNA and the linker has already occurred at the end of translation andthis [rACCA-amino acid] substrate is connected to the mRNA on theribosome via the linker and shows a locally very high concentration.

The fact that the [mRNA]-[linker]-[peptide] complex thus formed on theribosome is stably kept even after the peptide chain dissociates fromthe ribosome is also supported in the Examples herein below.

4. Selection of a Peptide Aptamer

In evolutionary molecular engineering, large amounts of potential genesare provided and clones having a target phenotype are selected from themin order to create a protein or peptide having a desired function orproperty.

Basically, a DNA population is prepared first to give an RNA populationas an in vitro transcript, and then a peptide population as an in vitrotranslation product. From this peptide population, a peptide having adesired function or property is selected by some screening system. Ifone wishes to obtain a peptide molecule binding to a specific protein,for example, the peptide population is injected into a targetprotein-immobilized column, whereby a mixture of peptide molecules boundto the column can be recovered. The template mRNA fused to each peptidemolecule like a tag in a population of the recovered peptide-mRNAcomplexes is converted back into the DNA by reverse transcriptase togive a biased library containing a lot of clones having a targetphenotype amplified by PCR, and then similar selection experiments areperformed again. Alternatively, it is also possible to perform reversetranscription reaction before selection for the purpose of makingnucleic acid moieties double-stranded in order to avoid the possibilityof recovering an RNA aptamer. By repeating this procedure, clones havinga desired phenotype become concentrated in the population overgenerations.

To identify a peptide aptamer, the gene for a peptide aptamer binding toa target substance can be cloned by repeating the steps of mixing alibrary of mapped molecules and the target substance, selecting mappedmolecules presenting peptides bound to the target substance (activespecies), and preparing a nucleic acid library by PCR from nucleic acidmoieties of the mapped molecules selected. The step of selecting mappedmolecules bound to the target substance can be accomplished by allowing[RNA (or DNA/RNA hybrid)]-[linker]-[peptide] complexes to bind to thetarget substance and separating them from other complexes by anappropriate method to identify a peptide having a desired bindingproperty.

The target substance may be a protein, nucleic acid, carbohydrate, lipidor any other compound. It is convenient to derivatize the targetsubstance with a label isolatable by binding to a solid phase in orderto separate active species complexes binding to the target substancefrom other complexes. For example, the target substance is biotinylatedand isolated by specific binding to an immobilized biotin-bindingprotein in the Examples herein below. Such specific binding pairs thatcan be used include, but are not limited to, biotin-binding protein(avidin, streptavidin, etc.)/biotin pairs as well as maltose-bindingprotein/maltose, polyhistidine peptide/metal ion (nickel, cobalt, etc.),glutathione-S-transferase/glutathione, antibody/antigen (epitope), etc.

By using evolutionary molecular engineering, it is possible in principleto obtain a peptide having a non-naturally occurring amino acid sequencefrom a gene library of DNA sequences consisting of randomly connectedfour bases A, T, G, C. Further, a unusual peptide containing anon-proteinogenic amino acid (or hydroxy acid) can also be synthesizedby translation as an in vitro translation product by introducing a tRNAacylated with the non-proteinogenic amino acid (or hydroxy acid) into atranslation system. A library of unusual peptides can be efficientlyobtained by repeating the steps of selecting an active speciespresenting a peptide having a desired binding property from a populationof complexes of a unusual peptide and an mRNA (or cDNA), amplifying amapped gene moiety and translating it again.

For details of molecular biology techniques with respect to thedescription herein above and below in the Examples, see, for example,Sambrook, Molecular Cloning: A Laboratory Manual, 3rd edition, ColdSpring Harbor Laboratory Press, 2001; Golemis, Protein-ProteinInteractions: A Molecular Cloning Manual, 2nd edition, Cold SpringLaboratory Press, 2005, etc.

The following examples further illustrate the present invention.However, these examples are only for illustrating the present inventionbut should not be construed to limit the scope of the present invention.

Example 1

[Synthesis Of Linkers]

Chimeric DNA/RNA oligonucleotides comprising a DNA and a RNA connectedvia polyethylene glycol (5 units of Spacer18) were used as linkers.Various linkers were purchased from BEX (Tokyo). In the sequence shownbelow, *A and *C correspond to RNA, and SPC18 corresponds to Spacer18(hexaethylene glycol).

an21-ACOA: 5′-CCCGCCTCCCGCCCCCCGTCC-[SPC18]₅-A*-C*-C*-A*-3′

[Aminoacylation of the Linkers]

L-phenylalanine or β-L-alanine was attached to the 3′ end of the an21-ACCA linker molecule via an ester bond by a flexizyme-catalyzedreaction. The reaction product was identified by acrylamideelectrophoretic analysis of the purified reaction product solution underacidic conditions. If the bands derived from the linker molecule areaminoacylated, the mobility decreases. Thus, aminoacylation efficiencycan be determined by comparing the intensity of bands derived fromunreacted materials and bands derived from the reaction product.

Acylation reaction for attaching L-phenylalanine was performed by adding5 μL of 20 μM flexizyme eFx, 20 μM an 21-ACCA linker, and a substrate(L-phenylalanine cyanomethyl ester) to 20% dimethyl sulfoxide in 0.1 MHEPES-potassium buffer (pH 7.5), 600 mM magnesium chloride, and reactingthe mixture on ice for 2 hours. Specifically, 40 μM linker moleculedissolved in pure water and flexizyme eFx (200 μM, 0.5 μL) were firstadded to 0.2 M HEPES-potassium buffer (pH 7.5), and the mixture washeated on a thermoblock (ND-MD1, Nissin Scientific Corporation) at 95°C. for 2 minutes, and allowed to stand at room temperature for 5minutes. Then, acylation reaction of the linker molecule was started byadding magnesium chloride (3 M, 1 μL) and a substrate (25 mM in dimethylsulfoxide, 1 μL) on ice, and the mixture was allowed to stand on ice for2 hours. For attaching β-L-alanine, β-L-alanine p-chlorobenzyl thioetherwas used as a substrate under the same conditions at pH 8.0. Thereaction was quenched by adding 40 μL of 0.3 M sodium acetate (pH 5.0).The reaction product was precipitated with ethanol, and the pellet waswashed with 70% ethanol and dissolved in 10 μL of 1 mM sodium acetate.After the reaction, the solution was separated by 20% denaturingpolyacrylamide gel electrophoresis (50 mM sodium acetate (pH 5.0), 6 Murea) under acidic conditions, and the gel after migration was analyzedby fluorescent staining with SYBR Green II (Invitrogen, SYBR is aregistered trademark of Molecular Probes Inc.).

As shown in FIG. 3, the results demonstrated that the mobility of thelinker molecule of the reaction product is lower than that of theunreacted control linker molecule, indicating that the linker moleculehas been aminoacylated by a flexizyme-catalyzed reaction.

[Synthesis of cDNAs]

The cDNAs used for forming peptide-mRNA complexes were prepared byannealing synthetic oligonucleotides by PCR. Synthetic DNAs having thesequences shown below were purchased from Operon Biotechnologies, Inc.(Tokyo). Each cDNA obtained by annealing these oligo DNAs comprises a T7promoter sequence, a ribosome binding site, a start codon, a peptideaptamer sequence, a spacer peptide sequence (CGSGSGS), an amber codonand a linker hybridization sequence.

TNF-a_D-Trp.R66: GCCGCTGCCGCTGCCGCAATGCTTCAGATACAGACAATGCAGACGTTGCATATGTATATCTCCTTC EMP1SS.F63:GAAGGAGATATACATATGGCAGCAGGTGGTACCTATTCTTCTCATTTTGG TCCGCTGACCTGGEMP1SS.R63: GCCGCTGCCGCTGCCGCATGCTGCACCACCTTGCGGCTTAGAAACCCAGGTCAGCGGACCAAA T7g10M.F48:AATACGACTCACTATAGGGTTAACTTTAAGAAGGAGATATACATATG CGS3an13.R39:TTTCCGCCCCCCGTCCTAGCTGCCGCTGCCGCTGCCGCA CGS3an21.R44:CCGCCTCCCGCCCCCCGTCCTAGCTGCCGCTGCCGCTGCCGCA

Annealing by PCR was performed by the following procedure.

20 uL of a PCR reaction solution (10 mM Tris-HCl (pH 9.0), 50 mMpotassium chloride, 2.5 mM magnesium chloride, 250 μM dNTPs, 0.2% TritonX-100 (Triton X-100, NACALAI TESQUE, INC.) and Taq polymerase)containing 250 nM T7g10M.F48 and TNF-a_D-Trp.R66 (or a combination ofEMP1SS.F63 and EMP1SS.R63) was prepared and reacted at 94° C. for 1 min,followed by 5 cycles of {50° C. 30 sec, 72° C. 30 sec} using a thermalcycler (TC-3000, Techne). Then, 100 μL of a PCR reaction solutioncontaining 500 nM each of the primers T7g10M.F48 and GCSan21.R44 (orGCSan13.R39) was prepared, and combined with 1 μL of the former PCRreaction product solution, and the mixture was reacted by temperaturecycling of 10 cycles of {94° C. 40 sec, 50° C. 40 sec, 72° C. 40 sec}.The reaction product was routinely purified by phenol-chloroformextraction, chloroform extraction, and ethanol precipitation.

[Preparation of mRNAs]

To prepare mRNAs, RNAs were synthesized by transcription reaction usingT7 RNA polymerase from the cDNA (TNF-α-DW (an 21)) prepared by theprocedure described above and the resulting reaction products wereroutinely purified by phenol-chloroform extraction, chloroformextraction, and 2-propanol precipitation. The purified mRNAs werediluted to a concentration of 10 μM as determined from the UV absorbanceat 260 nm. [Transcription/translation, hybridization with the linkersand conjugation to a peptidyl acceptor]

Translation from mRNAs and complex formation with linker molecules ortranscription from cDNAs, translation and complex formation with linkermolecules were performed using a reconstituted cell-free translationsystem.

The reconstituted cell-free translation system used in the presentexample comprises the following biological polymers: 70S ribosome (1.2uM), translation initiation factors (IF1 (0.7 uM), IF2 (0.4 uM), IF3(1.5 uM)), elongation factors (EF-G (0.26 uM), EF-Tu/EF-Ts complex (10uM)), translation termination factors (RF2 (0.25 uM), RF3 (0.17 uM)),methionyl-tRNA transformylase (MTF (0.6 uM)), ribosome recycling factor(RRF (0.5 uM)), aminoacyl-tRNA synthetases (AlaRS (0.73 uM), ArgRS (0.03uM), AsnRS (0.38 uM), CysRS (0.02 uM), GlnRS (0.06 uM), GluRS (0.23 uM),GlyRS (0.09 uM), HisRS (0.02 uM), IleRS (0.4 uM), LeuRS (0.04 uM), MetRS(0.03 uM), PheRS (0.68 uM), ProRS (0.16 uM), SerRS (0.04 uM), ThrRS(0.09 uM), TrpRS (0.03 uM), ValRS (0.02 uM), AspRS (0.13 uM), LysRS(0.11 uM), TyrRS (0.02 uM)), creatine kinase (CK, from Roche (4 ug/mL)),myokinase (MK, from Roche (3 ug/mL)), pyrophosphatase (PPa (0.1 uM)),nucleotide diphosphate kinase (NDK (0.1 uM)), T7 RNA polymerase (17phage gene-derived recombinant, 0.1 uM), E. coli tRNA (from Roche, 1.5mg/mL). The ribosome was purified from E. coli in the logarithmic growthphase, while various proteins other than the ribosome are recombinantproteins expressed and purified from cloned genes for E. coli, unlessotherwise specified.

Besides the biological polymers, the following components are included:50 mM HEPES-KOH (pH7.6), 2 mM NTPs, 20 mM creatine phosphate, 100 mMpotassium acetate, 2 mM spermidine, 1 mM dithiothreitol, 6 mM magnesiumacetate, 0.1 mM 10-formyl-5,6,7,8-tetrahydrofolic acid.

2.5 μL of a transcription/translation reaction solution was prepared,containing 1.5 μM mRNA or 0.15 μM cDNA, an aminoacyl-initiator tRNAbearing N-chloroacetyl-D-tryptophan (CIAc-D-Trp) as an acyl group(prepared by the procedure disclosed in JPA-2008-125396), and 19 aminoacids (each 5 mM) constituting natural proteins except for methionine inaddition to the components mentioned above.

In the case where an mRNA is preliminarily prepared by transcriptionreaction and then translated into a peptide and the resulting peptide isfused to a linker, a peptide-mRNA complex was prepared by the followingprocedure. First, 4 mM sodium acetate, pH 5.0 and mRNA were mixed in1:3, and the mixture was heated on a thermoblock at 95° C. for 1 minuteand then allowed to stand at room temperature for 5 min. A 25 μM linkersolution dissolved in 1 mM sodium acetate was added and the mixture wasallowed to stand for 10 minutes to form an mRNA-linker complex. To thiswere added the other components of the translation system and themixture was incubated in a constant-temperature water bath (NT-202D,Nissin Scientific Corporation) at 37° C. for 30 minutes, then at roomtemperature for 12 min, and 0.1 M EDTA (ethylenediaminetetraacetic acid,molecular biology grade, NACALAI TESQUE, INC.) adjusted to pH 7.5 wasadded at a final concentration of 20 mM, and the mixture was furtherincubated in the constant-temperature water bath at 37° C. for 30minutes.

In the case where a cDNA is added to a reaction solution andtranscription, translation and fusion to a linker are performed in thisreaction solution, a peptide-mRNA complex was prepared by the followingprocedure. First, a solution containing the components other than thelinker was prepared and reacted in a constant-temperature water bath at37° C. for 30 minutes to perform transcription and translationreactions. To this was added 0.25 μL of 25 μM linker solution, and themixture was further incubated at 37° C. for 30 minutes, then at roomtemperature for 12 minutes. Then, 0.1 M EDTA (ethylenediaminetetraaceticacid, molecular biology grade, NACALAI TESQUE, INC.) adjusted to pH 7.5was added at a final concentration of 20 mM, and the mixture was furtherincubated in the constant-temperature water bath at 37° C. for 30minutes.

[Reverse Transcription]

To improve stability of the mRNA moiety of the peptide-mRNA complex, anRNA-DNA hybrid strand was formed by reverse transcription reaction.Specifically, the following procedure was applied. To the translationreaction product described above were added 12 mM Tris-HCl (pH 8.3), 5μM reverse transcription primer (CGS3an 13.R21), 0.5 mM dNTPs, 18 mM Mg(OAc)₂, and 10 mM KOH (each expressed by the final concentration). Tothis was added 1 unit of M-MLV Reverse Transcriptase (RNaseH Minus,Point Mutant, Promega), and the mixture was reacted in aconstant-temperature water bath at 42° C. for 10 minutes. Then, EDTA andhydrochloric acid were added at final concentrations of 10 mM and 18 mM,respectively.

[Selection]

The following procedure was taken to assess whether or not thepeptide-mRNA complex molecule prepared in this manner is recovered byinteraction between the presented peptide and a target protein.

Human tumor necrosis factor-alpha (hereinafter referred to as TNF-α) waschosen as a target protein.

To 6 μL of a cell-free translation reaction product solution containinga peptide-mRNA complex molecule was added biotinylated TNF-α protein ata final concentration of 250 nM, and this solution was transferred to a0.6 mL ultra-low retention Eppendorf tube (platinum (ultra-lowretention) tube, BM Equipment Co., Ltd.), and gently stirred on arotator (RT-30 mini, Tietech Co., Ltd.) at 4° C. for 1 hr to inducebinding. To this solution was added 3 μL of a suspension ofstreptavidin-immobilized magnetic beads (Dynabeads® Streptavidin M-280,Invitrogen), and the mixture was stirred for further 10 minutes. Then,the magnetic beads were separated by centrifugation and using a magnetholder, and the supernatant was removed, and the pellet was resuspendedin 50 μL of TBS (Tris Buffered Saline, 50 mM Tris-HCl(trishydroxymethylaminomethane, NACALAI TESQUE, INC.) pH 7.5, 150 mMsodium chloride) containing 0.05% Tween20 (polyoxyethylene sorbitanmonolaurate, NACALAI TESQUE, INC.). This washing process was repeatedfour times.

Subsequently, 25 μL of PCR (taq-) buffer (10 mM Tris-HCl (pH 9.0), 50 mMpotassium chloride, 2.5 mM magnesium chloride, 250 μM dNTPs, 0.25 μMT7g10m.F48, 0.25 μM CGS3an 13.R21, 0.2% Triton X-100) was added to themagnetic beads, and the suspension was heated on a thermoblock at 95° C.for 5 minutes and then the supernatant was collected, whereby a DNA wasrecovered from the magnetic beads after reverse transcription.

[Quantification by Real-Time PCR]

The amount of the DNA was determined by real-time PCR before the targetprotein was added and after it was recovered from the magnetic beads.Using LightCycler® 1.5 (Roche Applied Science) as a real-time PCRsystem, a reaction solution containing Taq polymerase, SYBR® Green I(1:100,000 dilution, Invitrogen), and a test sample solution in the PCR(taq-) buffer described above was assayed.

Model Experiment Targeting Tnf-α

TNF-α and a peptide aptamer binding to TNF-α, TNF-α-DW were chosen as atarget protein and a peptide aptamer used to test the function of thelinkers.

The TNF-α protein used in the present example was a recombinant solubleTNF-α expressed by E. coli. The recombinant TNF-α is a fusion of asequence of the 77th to 233rd amino acids of wild-type TNF-α to AviTagsequence (GLNDIFEAQKIEWHE) and His×6 tag sequence at the N-terminus.This protein and a biotin ligase (BirA) using AviTag as a substrate areco-expressed in E. coli, whereby the side chain of the lysine residue ofAviTag is biotinylated, so that the TNF-α used in the present examplecan be readily separated by streptavidin-immobilized beads.

The TNF-α-DW used as a peptide aptamer was a peptide XQRLHCLYLKH(X:Ac-D-Trp) circularized with a thioether formed by a reaction between thechloroacetyl group of a non-proteinogenic amino acidN-chloroacetyl-D-tryptophan (ClAc-D-Trp) and the thiol group in the sidechain of cysteine in the peptide sequence. A cDNA encoding a sequencecontaining a spacer amino acid sequence CGSGSGS fused to the C-terminusof this peptide was prepared by the method described above to form apeptide-mRNA complex molecule in a cell-free transcription/translationsystem.

The yields of peptide-mRNA complexes using a combination of this peptideaptamer and the target protein are shown in FIG. 4 and FIG. 5. Theyields with an 21-Phe, that is a linker having L-phenylalanine attachedto the 3′ end via an ester and an 21-β-Ala were 1.30% and 0.25% when themRNA was added to the translation reaction solution (FIG. 4), or 0.65%and 0.38% when a cDNA was added (FIG. 5). However, the yield with the an21-ACCA linker unmodified at the 3′ end was about several tens of timeslower than those obtained with the modified linkers (0.015% in the caseof mRNA, or 0.007% in the case of cDNA), showing that peptide-mRNAcomplex molecules are efficiently recovered by conjugatingL-phenylalanine or β-L-alanine to the C-terminus of the peptidesynthesized by translation via a covalent bond.

Example 2

Verification of Mapping Between the Presented Peptide and the mRNA

In the foregoing experiments using a single type of the presentedpeptide and the mRNA encoding its sequence, one cannot exclude thepossibility that another linker molecule binds to the peptidesynthesized by translation from the mRNA as an acceptor or thepossibility that the linker molecule and mRNA molecule forming a doublestrand are replaced by another linker molecule or mRNA during themanipulation. Thus, the following experiments were performed to verifythat mapping between the peptide presented by recoveredpeptide-mRNA-linker complex molecules and the mRNA has been exactlymade.

First, an mRNA or cDNA encoding a peptide sequence that does not bind tothe target protein (EMP1SS: XAAGGTYSSHFGPLTWVSKPQGGAA, wherein X isAc-D-Trp similarly to TNF-α-DW) as a control of TNF-α-DW was prepared bythe procedure shown in Example 1, and mixed with an mRNA or cDNAencoding TNF-α-DW in a molar ratio of 100:1. This mixture was used toperform experiments similar to those described above, and the recoveredDNA was amplified by PCR under the following conditions. That is, 20 μLof a reaction solution containing Taq polymerase and a test samplesolution in PCR (taq-) buffer was prepared and reacted by temperaturecycling of 30 cycles of {94° C. 40 sec, 61° C. 40 sec, 72° C. 40 sec}(with a slope of 0.5° C./sec from 61° C. to 72° C.) using a thermalcycler. 2.5 μL of the reaction product solution was separated byelectrophoresis using a gel consisting of TAE (40 mM Tris-acetate, 1 mMEDTA) and 3% agarose (low electroendosmosis, NACALAI TESQUE, INC.), andelectrophoretograms were obtained under a transilluminator afterstaining with ethidium bromide. The results are shown in FIG. 6.

A band derived from the mRNA encoding TNF-α-DW is observed at theposition of about 90 bp, while a band derived from the mRNA encoding thecontrol EMP1SS is observed at the position of about 150 bp. When the an21-ACCA linker containing no peptidyl acceptor was used, only a bandderived from the mRNA of EMP1SS was observed, reflecting the molar ratioof the two types of cDNA in the initial mixture. With an 21-Phe,however, a band derived from the mRNA of TNF-α-DW was observed with afluorescent intensity comparable to or higher than that of EMP1SS. Thus,it was demonstrated that peptide-mRNA complexes presenting TNF-α-DW thatbinds to the target protein were recovered more efficiently thanpeptide-mRNA complexes presenting EMP1SS that does not bind to thetarget. This indicates that mapping between the peptide presented bypeptide-mRNA-linker complex molecules and the mRNA does not changeduring the experiments. In addition, this result was equally obtainedeither when peptide-mRNA complexes were formed by adding the mRNAs intothe translation reaction solution or peptide-mRNA complexes were formedby adding cDNAs.

Example 3

Selection of a TNF-α-DW-Spiked Library

[Random Peptide Library]

Evaluation was made to determine whether or not an mRNA or cDNA encodinga low copy number peptide aptamer contained in an mRNA library encodingrandom peptide sequences can be selected by repeating selection multipletimes.

First, a peptide aptamer library presenting a random sequence of 8-12amino acids was prepared. This library was prepared as an mRNA encodingthe random sequence. This library has a similar structure to those ofthe preparations described above except that the sequence regionpresenting TNF-α-DW or EMP1SS is randomized and the region with whichthe linker hybridizes to form a double strand has a length of 13 bp.

[Preparation of a TNF-α-DW-Spiked Library]

A spiked library was prepared by mixing an mRNA encoding TNF-α-DW(TNF-α-DW (an13): the region forming a double strand with the linker hasa length of 13 bp) with this random peptide library in a molar ratio of1,000,000:1.

[Round 1]

This spiked mRNA library and an 21-Phe linker was used to performtranslation and peptide-mRNA complex formation following the proceduresshown in Examples 1 and 2 above (round 1).

Negative selection was performed in which a peptide-mRNA complexsolution was mixed with unbound streptavidin-immobilized magnetic beadsat 4° C. for 10 minutes, whereby peptide-mRNA complexes binding to themagnetic beads were eliminated. To this solution was added TNF-α at afinal concentration of 250 nM, and mixed at 4° C. for 1 hour.Streptavidin-immobilized magnetic beads were added to this solution, andbound at 4° C. for 10 minutes and washed with TBST four times.

Then, the washed magnetic beads were suspended in 10 μL of a reversetranscription reaction solution (5 units/μL M-MLV Reverse Transcriptase(Promega), 2 μM CGS3an 21.R44, 0.5 mM dNTP, 10 mM Tris-HCl (pH 8.3), 15mM potassium chloride, 0.6° C. mM magnesium chloride, 2 mMdithiothreitol), and the suspension was warmed at 42° C. for 1 hr toperform reverse transcription reaction. Subsequently, 15 μL of PCR(taq-) buffer (containing 0.25 μM T7g10m.F48 and 0.25 μM CGS3an 21.R44as primers) was added to the suspension of the magnetic beads, and themixture was heated on a thermoblock at 95° C. for 5 minutes and then thesupernatant was collected, whereby a DNA was recovered from the magneticbeads after reverse transcription.

An aliquot each of the mRNA immediately after translation and therecovered DNA was assayed for the copy number by real-time PCR in thesame manner as in Example 1. Further, amplification by PCR was performedin the same manner as in Example 2 and terminated before theamplification reaction reached saturation to avoid reannealing of thereaction products to each other.

[Rounds 2, 3 and 4]

In round 2 and the subsequent rounds, the results were compared in thecase where an mRNA synthesized from the PCR product of the previousround was used for translation reaction or the cDNA obtained as the PCRproduct of the previous round was used to perform transcription andtranslation reactions at the same time. Experimental procedures weresimilar to those of Examples 1 and 2 except that negative selection wasperformed multiple times. Specifically, the number of times of negativeselection increases by one in each round. In round 2 and the subsequentrounds, PCR amplification was also terminated before the amplificationreaction reached saturation.

[Identification of Selected Sequences]

After the operation of round 2 was repeated multiple times, a greatincrease in binding affinity was observed in round 4. Changes in bindingaffinity with the number of rounds are shown in FIG. 7. The PCR productswere routinely cloned using pGEM-T easy Vector System I (Promega), and 5clones each were analyzed by DNA sequencing. The results showed that allthe resulting clones had the same sequence as that of TNF-α-DW (an21).

It was demonstrated that peptide aptamers can be selected from peptidelibraries consisting of random sequences by this method.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: Synthetic oligonucleotide

SEQ ID NO: 2: Synthetic oligonucleotide

SEQ ID NO: 3: Flexizyme Fx

SEQ ID NO: 4: Flexizyme eFX

SEQ ID NO: 5: Flexizyme dFx

SEQ ID NO: 6: Synthetic oligonucleotide

SEQ ID NO: 7: Synthetic oligonucleotide

SEQ ID NO: 8: Synthetic oligonucleotide TNF-a_D-Trp.R66

SEQ ID NO: 9: Synthetic oligonucleotide EMP1SS.F63

SEQ ID NO: 10: Synthetic oligonucleotide EMP1SS.R63

SEQ ID NO: 11: Synthetic oligonucleotide T7g10M.F48

SEQ ID NO: 12: Synthetic oligonucleotide CGS3an 13.R39

SEQ ID NO: 13: Synthetic oligonucleotide CGS3an 21.R44

SEQ ID NO: 14: Synthetic oligonucleotide CGSan13.R21

SEQ ID NO: 15: TNF-alpha-DW (an 21)

SEQ ID NO: 16: TNF-alpha-DW (an 13)

SEQ ID NO: 17: EMP1SS (an 21)

SEQ ID NO: 18: TNF-alpha

SEQ ID NO: 19: Flexizyme aFx.

(Notes) In the DNAs of SEQ ID NOs: 15-17, the codon for methionine isassigned to a non-proteinogenic amino acid (ClAc-D-Trp) (genetic codereprogramming).

1. A linker used for preparing a conjugate in which an mRNA and apeptide as a translation product thereof are coupled via the linker in areconstituted in vitro protein synthesis system, said linker comprising:a single-stranded structure region having side chain bases pairing withthe bases at the 3′-end of the mRNA at one end of the linker, and apeptidyl acceptor region having a group capable of binding to thetranslation product by peptidyl transfer reaction at the other end ofthe linker, wherein the peptidyl acceptor region has a structurecontaining an amino acid attached to an oligo RNA consisting of anucleotide sequence of ACCA via an ester bond; and said ester bond isformed by an aminoacylation reaction using an artificial RNA catalyst.2. The linker of claim 1 wherein the single-stranded structure regionand the peptidyl acceptor region are connected via a polyethylene glycolmoiety.
 3. The linker of claim 1 or 2 wherein the single-strandedstructure region consists of a single-stranded DNA.
 4. The linker ofclaim 1 wherein the artificial RNA catalyst used in the aminoacylationreaction has a chemical structure consisting of any one of the RNAsequences below: (SEQ ID NO: 3)GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU (SEQ ID NO: 4)GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU (SEQ ID NO: 5)GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGGU (SEQ ID NO: 19)GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGGU.


5. A process for preparing an [mRNA]-[linker]-[peptide] conjugate inwhich an mRNA and a peptide as a translation product thereof are coupledvia the linker of claim 1, comprising the steps of: preparing the linkerof claim 1, synthesizing an mRNA having a sequence capable ofhybridizing with the base sequence of the single-stranded structureregion of the linker downstream of a sequence encoding a peptide; andcontacting the linker with the mRNA and translating the mRNA into thepeptide in a reconstituted in vitro protein synthesis reaction solution.6. A process for preparing an [mRNA]-[linker]-[peptide] conjugate inwhich an mRNA and a peptide as a translation product thereof are coupledvia the linker of claim 1, comprising the steps of: preparing the linkerof claim 1, synthesizing a template DNA for an mRNA having a sequencecapable of hybridizing with the base sequence of the single-strandedstructure region of the linker downstream of a sequence encoding apeptide, and introducing the linker and the DNA into a reconstituted invitro protein synthesis reaction solution, thereby performingtranscription from the DNA into the mRNA and translation into thepeptide as well as complex formation between the linker and the mRNA. 7.The process of claim 5 or 6 wherein the reconstituted in vitro proteinsynthesis reaction solution contains a tRNA charged with anon-proteinogenic amino acid or hydroxy acid, whereby the translatedpeptide constitutes a unusual peptide.
 8. A library comprising[mRNA]-[linker]-[unusual peptide] conjugates prepared by the process ofclaim
 7. 9. A method for selecting a peptide aptamer that binds to atarget substance from a library of [mRNA]-[linker]-[peptide] conjugatesin which each mRNA and a peptide as a translation product thereof arecoupled via the linker of claim 1, said method comprising the steps of:preparing the linker of claim 1; preparing an mRNA library comprisingmRNAs each having a sequence capable of hybridizing with the basesequence of the single-stranded structure region of the linkerdownstream of a sequence encoding a random peptide sequence; contactingthe linker with the mRNA library and performing translation into thepeptide in a reconstituted in vitro protein synthesis reaction solution,thereby preparing an [mRNA]-[linker]-[peptide] conjugate library;contacting the target substance with the [mRNA]-[linker]-[peptide]conjugate library; and selecting a conjugate presenting the peptidebound to the target substance.
 10. The method of claim 9 wherein thetarget substance has been biotinylated.
 11. The method of claim 9 or 10wherein the reconstituted in vitro protein synthesis reaction solutioncontains a tRNA charged with a non-proteinogenic amino acid or hydroxyacid, whereby the translated peptide constitutes a unusual peptide. 12.A process for preparing the linker of claim 2, comprising the steps of:synthesizing a chimeric oligonucleotide consisting of thesingle-stranded structure region and an oligo RNA of a sequence of ACCAconnected via a polyethylene glycol moiety; and attaching an amino acidto adenosine at the 3′ end of the chimeric oligonucleotide via an esterbond by a reaction using an artificial RNA catalyst, thereby preparing alinker consisting of the single-stranded structure region and thepeptidyl acceptor region connected via the polyethylene glycol moiety.13. The method of claim 12 wherein the artificial RNA catalyst has achemical structure consisting of any one of the RNA sequences below:(SEQ ID NO: 3) GGAUCGAAAGAUUUCCGCAGGCCCGAAAGGGUAUUGGCGUUAGGU(SEQ ID NO: 4) GGAUCGAAAGAUUUCCGCGGCCCCGAAAGGGGAUUAGCGUUAGGU(SEQ ID NO: 5) GGAUCGAAAGAUUUCCGCAUCCCCGAAAGGGUACAUGGCGUUAGGU(SEQ ID NO: 19) GGAUCGAAAGAUUUCCGCACCCCCGAAAGGGGUAAGUGGCGUUAGGU.